The biochemical mechanism by which botulinum neurotoxins enter and act upon nerve cells is largely unkown. Part of the entry process involves unfolding the toxin protein followed by the passage of the linear toxin through a channel leading to the cytosol. We did the first direct study of this process using BoNT type A and E on cultured spinal cord neurons. We find that internalization of each toxin has a unique pH requirement. BoNT/A requires a greater degree of acidification than does BoNT/E. We also have found that both toxins require cytosolic calcium and protein kinase activity to become active. The aim is to do the first long-term recovery study for these two toxins, correlate their activity with neurotransmitter release and examine the pH, calcium and kinase requirements for each.